1. Field of the Invention
The present invention relates to isolated nucleotide sequences; methods for using the sequences to make transgenic plants which produce edible fruits having improved firmness and longevity; and vectors having the nucleotide sequences incorporated therein. More specifically, the present invention relates to transgenic plants and methods for making the same, the genomes of said plants having incorporated therein foreign nucleotide sequences which function to inhibit production of fruit ripening specific lipoxygenase (xe2x80x9cFRS-LOXxe2x80x9d) in a ripening fruit. The inhibition of FRS-LOX gene expression provides a mechanism to improve characteristics of a fruit, including controlling fruit senescence and tissue softening associated with post-maturation Processes.
2. Discussion of Related Art
Lipoxygenases (xe2x80x9cLOXsxe2x80x9d) are nonheme iron-containing dioxygenases that catalyze the incorporation of molecular oxygen into unsaturated fatty acids containing cis, cis 1,4-pentadiene structure to yield a 1-hydroperoxy-2-4-trans, cis pentadiene product. Typical substrates for LOXs in plants are linoleate (C18:2) and linolenate (C18:3) fatty acids, while animal LOXs prefer arachidonate (C20:4). Some LOXs are able to act on substituted fatty acid substrates, while others require free fatty acids. LOXs can vary in respect to (1) the site of primary hydrogen abstraction, (2) the direction of the double bond shifts in the primary radicle leading to the hydroperoxide product and (3) the stereospecificity of both hydrogen abstraction and dioxygen insertion (Ford-Hutchinson et al., Annu. Rev. Biochem. 63:383, 1994).
In plants, most fatty acids are esterified to a glycerol backbone in the form of glycerolipids and lipases are thought to cleave these phosopholipids into usable plant LOX substrates (free fatty acids). The LOX-catalyzed fatty acid hydroperoxides serve as intermediates for a number of secondary reactions leading to jasmonic acid, traumatin, traumatic acid, volatile alcohols (hexanal), aldehydes, ketols and 9C oxo fatty acids (Vick and Zimmerman, Biochemistry of Plants, Vol. 9:53, 1987; Hildebrand, Physiol. Plantarum 76:249, 1989). The 5-LOX from humans is representative of a unique type of LOX that requires the association of the 5-LOX Activating Protein (FLAP) for activity. Also, a LOX from the rabbit reticulocyte has been reported to attack mitochondrial membranes in the absence of any lipid hydrolyzing enzymes during the maturation of erythroid cells (Schewe et al., Adv. Enzymol. 58:191, 1986).
LOXs have been found in a wide range of organisms including higher plants, animals, yeast, fungi and cyanobacterium (Siedow, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:145, 1991). LOX multigenic families have been characterized in several plant and animal species (Siedow, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:145, 1991; Ford-Hutchinson et al., Annu. Rev. Biochem. 63:383, 1994). The best characterized plant LOXs are the three soybean cotyledon LOX monomer isozymes; L-1, L-2 and L-3, all globular, water soluble proteins with MWs of about 96 kD. LOXs from rice, soybean, cotton, sunflower, tomato, Arabidopsis, cucumber, kiwi and tobacco are some that have been identified and are on the order of 95 kD, with the exception of pea (72-108 kD). Sequences reported for plant LOXs are approximately 60% homologous to one another. Human LOXs are about 60% homologous and are only 25% identical to plant LOXs (Ford-Hutchinson et al., Annu. Rev. Biochem. 63:383, 1994). Some of the plant LOXs are larger than animal LOXs and show homology with them only in limited regions (Minor et al., Biochem. 32:6320, 1993).
The function of various LOX isozymes in plant and mammalian systems is unknown. The hydroperoxide products from some animal LOXs serve as precursors in the production of leukotrienes and lipoxins, regulatory molecules involved in responses include leukotiene induced altered cell functions such as chemotaxis and chemokinesis. Roles of LOX during all stages of plant growth and development have been speculated (Siedow, Annu. Rev. Plant Physiol. Plant Mol. Bio. 42:145, 1991). LOX activity has been demonstrated in rapidly growing young tissues (germinating seedlings). It has been suggested that jasmonic acid and hydroperoxide free radicles, primary and secondary products of LOX, may play roles in plant senescence by promoting cell membrane deterioration, inhibiting protein synthesis and chloroplast photochemical activity. Upon tissue wounding, increases in LOX activity and mRNA accumulation have been seen in some plants. Traumatin and traumatic acid may be involved in wound healing of damaged plant tissues (Hildebrand, Physio. Plantarum 76:249, 1989). Another secondary LOX product, hexanal, may be produced in response to pest/pathogen attack. However, no evidence to support any of these hypotheses has been obtained.
Fruits and vegetables are highly perishable crops and significant losses often occur after their harvest but before they reach the consumer. The primary causes of these losses are the inability to control: 1) senescence of these crops; 2) the ripening process in fruits; and 3) ripening-and sensescence-associated tissue softening. One of the objectives of plant breeders for many years has been to control these processes by introducing traits from wild germ plasm into the cultivated species. In recent years, attempts have been made to use recombinant DNA technology to modify some of these traits. Both antisense and co-suppression technologies have been used in some crop plants to modify the expression of specific genes which may have deleterious effects on plant growth and development or crop productivity in general. However, none have proven fully satisfactory in reducing the rate of tissue softening associated with post-maturation plant processes.
Major transitions in fruit development and metabolism accompany the initiation of fruit ripening. In addition to alterations in pigment biosynthesis and production of volatile compounds, fruits undergo significant changes in texture during ripening. The biochemical bases for ripening- and senescence-associated fruit softening are not yet understood; however, dissolution of the middle lamella and cell wall separation due to depolymerization of pectins by polygalacturonase as well as loss of calcium have been suggested to contribute to fruit softening. Only slight improvement in fruit integrity has been reported in fruits with low polygalacturonase activity (Schuch et al. HortScience 26: 1517, 1991; Carrington et al., Plant Physiology 103: 429, 1993).
There is a need for transgenic plants which produce fruits having modified ripening and post-maturation characteristics including improved quality and texture, greater firmness, longer shelf life, better packing and storage characteristics and improved processing characteristics.
The invention described herein features inhibiting the expression of fruit ripening specific lipoxygenase (xe2x80x9cFRS-LOXxe2x80x9d) in a plant, specifically in cells of a fruit of the plant. For instance, foreign DNA can be introduced into cells to reduce FRS-LOX production. In a preferred embodiment, the cell is a plant cell and the genetic material is a sense or antisense fragment substantially similar to a portion of the FRS-LOX cDNA shown in SEQ ID NO:4. Most preferably, the cell is a cell of a fruit-bearing plant, such as a tomato plant cell.
It is expected that the present invention can be applied to the inhibition of FRS-LOX gene products in a wide variety of useful plants. These may include, for example, commercially important fruit-bearing plants in which post-maturation weakening reduces economic value, such as melons, peaches, bananas, apples, strawberries, kiwi fruit, and in particular the tomato.
The present inventors have made the surprising discoveries that (1) inhibition of FRS-LOX greatly improves fruit qualities such as, for example, firmness and shelf life; and (2) antisense and co-suppression (sense) technologies can be successfully used to inhibit FRS-LOX gene expression in plants so as to provide fruits having superior qualities such as, for example, firmness and shelf life. It is believed that improved fruit qualities result from reduced activity of degradative pathways (e.g., membrane deterioration); however, it is not intended that the present invention be limited to this theory.
Briefly describing one aspect of the present invention, there is provided a method for making a transformed plant having improved fruit quality comprising inserting a nucleotide sequence into DNA of the plant in a sense or antisense orientation under a suitable promoter so as to inhibit production of lipoxygenase in the fruit of the plant as it ripens. In a preferred method, a sequence of nucleotides is inserted into a target cell by providing a vector comprising the nucleotide sequence and contacting the vector with the target cell.
Additional aspects of the present invention include vectors having incorporated therein nucleotide sequences having substantial similarity to all or a portion of the sequence of SEQ ID NO:4(FIG. 4).
Additional aspects of the invention include constructs selected from the group consisting of pMLSL, PMLAL, pUSL2, pUAL2, pUEL300S and pUEL300A, which are useful for inserting foreign DNA into a plant cell genome.
According to other aspects of the present invention there are provided transgenic fruits, transgenic plants and transgenic host cells, preferably plant cells such as germ cells and cotyledon cells. These various transgenic hosts are preferably transformed by having incorporated into their genomes, nucleotide sequences as delineated above and described in greater detail below.
It is an object of the present invention to provide transgenic plants which produce fruits having modified ripening characteristics, including improved quality and texture, greater firmness, longer shelf life, better packaging and storage characteristics and improved processing characteristics.
Further objects, advantages, and features of the present invention will be apparent from the detailed description which follows.